Slow-growth conservation- cryopreservation and Analysis of genetic stability of in vitro regenerated Coelogyne Nervosa A.Rich A endemic orchid

Abstract

Coelogyne Nervosa A. Rich., an endangered medicinal epiphyte (lithophyte) is a strict endemic of the Western Ghats distributed across Karnataka, Kerala and Tamil Nadu. KC medium supplemented with 2,4-D is best suited for direct embryo induction from 80 days old protocorms. Hormone-free KC basal medium supported the conversion of somatic embryos into complete plantlets. For the medium-term storage experiment, somatic embryos were placed on storage media containing various growth inhibitors (such as different levels of chlormequat, maleic hydrazide and paclobutrazol). They were stored for ten months to assess the percentage of survival. The surviving shoots were transferred to the recovery medium for eight weeks. At 8°C, the highest survival rate was observed with ABA at 0.2 mg L−1. For cryopreservation: droplet-vitrification best results were obtained when the somatic embryos, after 20 days of subculture were immersed in an osmoprotectant solution. Osmo-protected samples were immersed in PVS2 (15% ethylene glycol and 15% DMSO) at 0°C for 30 min. Pre-prepared Somatic embryos were plunged into cryovials for one day. Regrowth levels were as high as 70%. DNA samples extracted from leaves of the mother plant, somatic embryos without storage, regenerates of slow growth preservation and cryopreservation. The samples were analysed by DNA based molecular marker – inter simple sequence repeats (ISSR) and found no genetic changes during the cryopreservation process.